Low DNA input for exome sequencing

[ymc]

I noticed that Agilent SureSelect has a low input protocol that only requires 200ng gDNA. Nextera also has an exome kit that claims to need only 50ng. Anyone with experience about them? Thanks!

[ElTony]

I used the sureselectXT for 200ng and it works very well.

[CCBIO]

And what about TruSeq CA? have you ever tried to use less than 50ng? (that is the amount of DNA the kit requires)
I would like to sequence some "precious" samples that, unfortunately, have very low concentrations and volumes. Anyone has tips? Thanks in advance!

[nucacidhunter]

Would you elaborate on your precious sample and wether it is RNA or DNA and estimated total amount? Do you want to prep a library and sequence it straight or would you want to do a sequence capture first?

[CCBIO]

Well, it is gDNA and the range is 5-10 ng (total) with a concentration of around 1 ng/ul. I was wondering if this could be enough either to perform a sequence capture or TruSeq CA

[nucacidhunter]

I am not aware of any kit for sequence capture using 5-10 ng DNA. As ymc has pointed the lowest input currently available are Nextera Rapid Capture and Agilent QXT kits requiring 50 ng DNA for sequence capture. The best option here seems to be library prep and direct sequencing. There are few low input DNA library prep kits including NEBNext Ultra DNA library prep kit (5-10 ng input), Ovation Ultralow Library (1-100 ng input), ThruPLEX FD kit (60 pg-50 ng input), and Accel-NGS 2S kit (10pg- 1ug input). They all claim to produce excellent libraries in their data sheet. Hopefully someone using these kits could share their experience in this forum.

[buckybadger]

I am not aware of any kit for sequence capture using 5-10 ng DNA. As ymc has pointed the lowest input currently available are Nextera Rapid Capture and Agilent QXT kits requiring 50 ng DNA for sequence capture.

I'm surprised to see the KAPA Biosystems Kits (KAPA Library Prep Kit; KAPA Hyper Prep Kit) not mentioned among these others. In my experience these kits generate very nice, high-complexity libraries across a broad range of gDNA input.

In regard to sequence capture, I've seen data using 100 ng and 10 ng gDNA input into the KAPA Library Prep Kit, followed by NimbleGen exome capture, producing very nice results. It's not posted anywhere I can find, but they will share the performance summary and raw reads if you ask a sales rep or tech support. I've also seen data on libraries constructed from as little as 1 ng gDNA input into the KAPA Hyper Prep Kit used in a NimbleGen panel capture with good results. I haven't asked yet, but they might have that data available too.

Some of the keys to good performance using low DNA input into library prep are: (1) getting the adapter-insert ratios right, or you might get too many unligated adapters/dimers that you'll have to remove with an additional AMPure step (0.7x Bead-DNA ratio works for me), and (2) choosing the right cycle number to amplify the library to just the amount that you need (but not too much more than that).

As you use decreasing amounts of DNA into library prep, but keep the amount of sequencing constant, the corresponding (and predictable) effect is a increase in the rate of PCR duplicates sequenced. This will be true whether you are doing straight library sequencing or if you insert a capture process into that overall workflow.

KAPA put on a nice webinar recently about the Hyper Prep Kit (particularly it's performance with poor quality FFPE samples) and it's available to DL or stream on their website here.

Cheers,

BB

[nucacidhunter]

Thank you buckybadger for sharing your experience. If KAPA has good products they should transparently make their data or a summary of results available online without any Hassel to whoever might be interested. I personally do not pursue products (even if I think they might be good) where supplier does not make their user guide or data sheet available online.

[buckybadger]

Hi Nucacidhunter. I can appreciate your perspective perfectly well, but it makes me wonder if I didn't give the wrong impression in my reply. My apologies if that's the case.

It's actually not difficult to get access to most of the information I was referring to; perhaps it is too recent to have been on the websites as white-papers, graphics, user manuals, etc, when you last looked?

For the newest Kapa Hyper Prep Kit data, here is a link to KAPA's webinar and a poster (KAPA/UWash/Ambry) from AGBT 2014 on their website. Sequence capture data is included in the webinar.

For the NimbleGen exome capture data using 100ng and 10ng of gDNA input into the KAPA Prep up front (again, this is the KAPA with-bead Prep kit, not the newer KAPA Hyper Prep kit), Roche tech support will set up an FTP transfer for you to download the raw reads and target files for analysis through your own pipeline. I think a lot of people here would prefer to look at the data that way anyway if they're serious about it. The relevant contacts to get to this are not easy to find, and I agree that part is a bit annoying, but I can save you from the trouble of playing email tag (and they are very helpful after you do get in touch with them). The email addresses are:

North America: [email protected]
Europe: [email protected]
Asia Pacific: [email protected]

If you have any problems getting to any of this data, please send me a private email and I will be happy to try to re-contact the people I dealt with directly, to do what I can to help.

Cheers,

BB